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HIV Cellular Pathogenesis II Benhur Lee, M.D. HIV Accessory Genes: Tat Rev Essential in vitro and in vivo Vif Essential in certain cell types (Permissive vs Non-permissive cells) Vpr Vpu Nef Non-essential in vitro, but leads to attenuated phenotype in vivo Nef • Major determinant of pathogenicity in vivo – nef-deleted SIV is severely attenuated in the rhesus macaque model – infection of macaques with recombinant SIV carrying a premature STOP codon (point mutation) results in rapid revertants with the nef ORF – Patients infected with nef-defective HIV have a dramatically decreased rate of disease progression (>15 years) – nef-deleted HIV do not deplete thymocytes as much, or replicate to as high titers, as wild-type HIV in the SCIDhu mice model Pleiotropic Functions of Nef N-myristoylation required for Nef activity--implies that membrane localization of nef is critical for its activity Consensus N-myristoylation Signal: MGxxx(S/T)(K/R)(K/R) MGxxx(S)(K)(K/R) HIV sequence Conservation in 99% 100% Nef protein: 100% ~50% Pleiotropic Functions of Nef • Down-regulates cell surface levels of CD4 • Down-regulates surface levels of major histocompatibility class I molecules • Mediates cellular signaling and activation • Enhances viral infectivity I. Down-modulation of surface CD4 • Down-modulation of surface CD4 via internalization followed by degradation via endosomal/lysosomal pathway • Advantages: – – • Prevents disadvantageous super-infection of host cell Enhance viral progeny release (by preventing Env-mediated sequestration of CD4 in secretory pathway) Evidence: – – – – Nef expression increases number of CD4 containing clathrin-coated pits Nef-induced CD4 down-modulation blocked by inhibitors of clathrin-coated pit-mediated endocytosis (e.g. ikaguramycin) Inhibition of lysosomal acidification (e.g. via chloroqine treatment) blocks Nef-induced CD4 degradation Expression of nef alone in T-cell lines can lead to CD4 downregulation (as determined by FACS) CD4 .. .................... ..................................... ... ........ ...................... ............................... . ............. ............... . ..... Nef-GFP I. Down-modulation of surface CD4 • Mechanism(s)? – Direct interaction with CD4 has not been biochemically demonstrable, but NMR analysis suggest a direct interaction with Kd ~0.87 mM; yeast two-hybrid assays also suggest an interaction – Acts as a connector to the host-cell endocytic machinery • Co-localizes with AP-2 on inner plasma membrane • Conserved dileucine based sorting motif (E/DxxxL ) in Nef is important for both CD4-downmodulation and AP-2 co-localization • Interacts with NBP-1 (identified through a yeast two-hybrid screen). NBP-1 is part of the vacuolar membrane ATPase complex in clathrin-coated pits (H subunit of vacuolar ATPase--VH1) • C-terminal diacidic motif (DD) in Nef is important for NBP-1 interaction, and, at least in SIV Nef, the dileucine motif is also important for NBP-1 interactions • ?? May bind to b-Cop, a coatamer protein which targets proteins to lysosomes NBP-1 II.Down-modulation of MHC Class I • Advantages: – Immune evasion; MHC Class I presents antigens to cytotoxic T- lymphocytes; alerts innate and adaptive immune system to virally infected cells • Evidence: – – – – Nef expression reduces susceptibility of HIVinfected cells to CTL mediated lysis in vitro selectively down-regulates only HLA-A and HLAB, which presents antigens to CTLs; does NOT down-regulate HLA-C and HLA-E, which inhibit NK-cell mediated cell lysis Thus, efficiency of CTL-mediated lysis (adaptive immunity) is reduced without increasing increasing susceptibility to NK cell lysis CTL HIV HIV antigen MHC Class I 51Cr 100% HIV Dnef HIV wt % Lysis E (Effector Cell) CTL 0% 1:2 1:5 1:10 E:T ratio 1:20 HIV antigen MHC Class I T (Target Cell) III. Mediates Cellular Activation and Signaling • Nef expression upregulates a transcriptional program that activates the HIV LTR (microarray analysis) III. Mediates Cellular Activation and Signaling • Nef expression upregulates a transcriptional program that activates the HIV LTR (microarray analysis) • Nef can induce secretion of paracrine factors that enhance viral replication; macrophage supernatants from cells transduced with nef-expressing adenoviral vector can facilitate HIV replication in resting lymphoid cultures Adv-nef supnt p24 (ng/ml) Adv-GFP supnt 3 6 9 (days) III. Mediates Cellular Activation and Signaling • Nef expression upregulates a transcriptional program that activates the HIV LTR (microarray analysis) • Nef can induce secretion of paracrine factors that enhance viral replication; macrophage supernatants from cells transduced with nef-expressing adenoviral vector can facilitate HIV replication in resting lymphoid cultures • Nef interacts with Pak2 (p21 activated kinase 2) and Nef/Pak2 complex may regulate many of Nef’s effect on gene transcription IV. Infectivity Enhancement • Magnitude of infectivity enhancement is allele dependent • Nef mediated enhancement can be provided in trans – reporter gene (e.g. GFP or luciferase) expression under control of the LTR promoter can be enhanced when nef expression vector is co-transfected • Mechanisms: – Increased RT activity; increased proviral DNA synthesis – Increased cytoplasmic delivery of viral particles Vif: Viral infectivity factor, required for robust replication only in certain cells HIV-1 ( HIV-1 (Dvif) Hut78, H9, 1º PBLs C8166, 293T, HeLa Permissive Non-permissive +++ replication +++ replication +++ replication no replication Two hypotheses: (a) Permissive cells express an activity (factor) that can compensate for vif. (b) Non-permissive cells have an inhibitory activity on viral replication, which is overcomed by vif. See Simon et. al., Nature Med. 4: 1397 Non-permissive wt Dvif Permissive wt Dvif Heterokaryon wt Dvif Which phenotype will dominate? Non-permissive: inhibitory cellular factor overcomed by vif ++ - ++ Infectivity ++ Permissive: compensatory factor similar to vif Denv vs Denv/Dvif Non-Permissive Permissive (ensures that all viruses are produced from Heterokaryons) Two hypotheses: (a) Permissive cells express an activity (factor) that can compensate for vif. (b) Non-permissive cells have an inhibitory activity on viral replication, which is overcomed by vif. Permissive Heterokaryon Non-permissive wt ++ Dvif - Permissive wt ++ Infectivity wt Dvif ++ - Dvif ++ Non-permissive: inhibitory cellular factor overcomed by vif Expression of CEM15 in CEM-SS (permissive cells) renders it non-permissive After subtractive-PCR for differentially expressed genes CEM15=APOBEC3G (Apolipoprotein B mRNA Editing enzyme, Catalytic polypeptide-like 3G) • APOBEC3G, cytosine deaminase – – APOBEC1, cytidine deaminase involved in mRNA editing AID (Activation-induced cytidine deaminase), involved in somatic hypermutation, required for immunoglobulin gene diversification • Incorporated into virions – Deaminates cytosines to uracils during first strand cDNA synthesis in target cells – Moderate sequence specificity Degradation: uracil in DNA recognized (Py-C-C) by host uracil DNA glycosidase, which removes uracil from DNA-->cleavage and degradation Hypermutation: massive C-->U conversion on minus strand leads to massive G-->A hypermutation of the plus strand; resultant provirus is so heavily mutated, no functional viral proteins are encoded How does vif overcome APOBEC3G’s antiviral function? • Reduces the level of APOBEC3G in producer cell – Expression of vif reduces APOBEC3G levels – Vif promotes degradation of APOBEC3G via Ubiquitinproteasome pathway – Vif impairs efficient translation of APOBEC3G mRNA